IL-7 fusion proteins

ABSTRACT

The invention is directed to a fusion protein which includes a first portion including an immunoglobulin (Ig) chain and a second portion including interleukin-7 (IL-7).

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 60/533,406, filed Dec. 30, 2003, the entire disclosureof which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to interleukin-7 (IL-7) fusion proteins, methodsof their production and uses thereof. The proteins of the invention areparticularly useful in treating disorders accompanied by immunedeficiencies and particularly diseases which involve T-celldeficiencies.

BACKGROUND OF THE INVENTION

A variety of disorders and therapies involve a deficiency of immunecells. For example, HIV infection results in a loss of CD4+ T-cells,while therapies such as chemotherapy and radiation therapy generallyresult in a loss of a wide variety of blood cells. Attempts have beenmade to provide specific protein drugs that can replenish specific typesof immune cells that are lost as a result of a disease or therapy. Forexample, in cancer chemotherapy, erythropoietin is used to replenish redblood cells, granulocyte colony-stimulating factor (G-CSF) is used toreplenish neutrophils, and granulocyte macrophage colony stimulatingfactor (GM-CSF) is used to replenish granulocytes and macrophages. Theseprotein drugs, although beneficial, have relatively short serumhalf-lives such that immune cell replenishment is often insufficient.Moreover, no specific treatment is currently in clinical use tospecifically stimulate T or B-cell development, even though loss ofthese cells as a result of disease or after certain myeloablativetreatments is know to be particularly deleterious to a patient's health.Thus, there exists a need in the art to develop immune systemstimulators and restoratives, particularly of lymphocytes, that haveextended serum half-lives.

SUMMARY OF THE INVENTION

The present invention is directed to interleukin-7 (IL-7) fusionproteins which have improved biological properties compared tocorresponding wild-type IL-7 proteins. Moreover, the present inventionis based, in part, on the finding that IL-7 fusion proteins havingparticular structural features have improved biological propertiescompared to wild-type recombinant IL-7.

Accordingly, in one aspect, the invention features a fusion proteinincluding a first portion comprising an immunoglobulin (Ig) chain and asecond portion comprising interleukin-7 (IL-7), wherein the IL-7 fusionprotein has an increased biological activity, such as an extended-serumhalf-life or in promoting the survival or expansion of immune cells, ascompared to wild-type IL-7.

In one embodiment, the invention features a fusion protein including afirst portion including an Ig chain and a second portion including IL-7,wherein the amino acid residues at positions 70 and 91 of IL-7 areglycosylated and the amino acid residue at position 116 of IL-7 isnon-glycosylated. Throughout this document, amino acid positions of IL-7refer to the corresponding positions in the mature, human IL-7 sequence.In one embodiment, the amino acid residue at position 116 of IL-7 isasparagine. In another embodiment, the amino acid residue at position116 of IL-7 is altered such that it does not serve as a glycosylationsite. In one embodiment, the IL-7 moiety comprises disulfide bondsbetween Cys2 and Cys92, Cys34 and Cys129, and Cys47 and Cys141 of IL-7.

In another embodiment, the invention includes a fusion protein includinga first portion including an Ig chain and a second portion includingIL-7, wherein the IL-7 comprises disulfide bonding between Cys2 andCys92, Cys34 and Cys129, and Cys47 and Cys141 of IL-7. In oneembodiment, the amino acid residue at position 116 of IL-7 isnon-glycosylated. In another embodiment, the amino acid residue atposition 116 of IL-7 is asparagine or is altered such that it does notserve as a glycosylation site. In another embodiment, the amino acidresidues at positions 70 and 91 of IL-7 are glycosylated.

The Ig chain is generally an intact antibody or portion thereof, such asan Fc region. The Ig chain of the IL-7 fusion protein can be derivedfrom any known Ig isotype and can include at least a portion of one ormore constant domains. For example, the constant domain can be selectedfrom the group consisting of a CH1 region, a hinge region, a CH2 region,and a CH3 region. In one embodiment, the Ig moiety includes a hingeregion, a CH2 region and a CH3 region. The Ig chain is optionallyconnected to the IL-7 portion by a linker.

Ig moieties of a single antibody isotype, such as IgG1 or IgG2, andhybrid Ig moieties are permitted in the present invention. For example,in one embodiment, the Ig moiety includes a hinge region derived fromone isotype (i.e. IgG2) and a CH region from another isotype (i.e.IgG1). An Ig chain including an Fc portion of IgG1 can advantageously bemodified to include the mutations Asn297Gln and Tyr296Ala. Furthermore,an Ig chain including an Fc portion of IgG2 can be advantageouslymodified to include the mutations Asn297Gln and Phe296Ala.

The IL-7 portion of the IL-7 fusion protein described above may comprisethe mature portion of the IL-7 portion. In one embodiment, the IL-7portion can further include a deletion, such as an internal deletion. Inone example, IL-7 can include an eighteen amino acid deletion of aminoacids 96 to 114 of SEQ ID NO:1.

In other embodiments, the invention includes purified nucleic acidsencoding the IL-7 fusion proteins described above and cultured hostcells including these nucleic acids.

In another aspect, the invention includes a method of preparing an IL-7fusion protein including expressing in a host cell the nucleic aciddescribed above and harvesting the fusion protein.

In another aspect, the invention includes a composition such as apharmaceutical composition which includes the fusion protein describedabove.

In another aspect, the invention includes a method of treating a patientby administering Fc-IL-7 fusion proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of human IL-7 (SEQ ID NO:1). Thesignal sequence is shown in bold. Also depicted in bold and italics is astretch of eighteen amino acids which can be deleted from the IL-7sequence.

FIG. 2 depicts the amino acid sequence of cow IL-7 (SEQ ID NO:2). Thesignal sequence is shown in bold.

FIG. 3 depicts the amino acid sequence of sheep IL-7 (SEQ ID NO:3). Thesignal sequence is shown in bold.

FIG. 4 depicts the amino acid sequence of mature human Fcγ1-IL-7 (SEQ IDNO:4).

FIG. 5 depicts the amino acid sequence of mature humanFcγ2(h)(FN>AQ)-IL-7 (SEQ ID NO:5).

FIG. 6 depicts the amino acid sequence of mature humanFcγ1(linker1)-IL-7 (SEQ ID NO:6).

FIG. 7 depicts the amino acid sequence of mature humanFcγ1(YN>AQ)(linker2)-IL-7 (SEQ ID NO:7).

FIG. 8 depicts the amino acid sequence of mature humanFcγ1(YN>AQ,d)(linker2)-IL-7 (SEQ ID NO:8).

FIG. 9 depicts the nucleic acid sequence for the Fc region of humanFcγ1-IL-7 (SEQ ID NO:22).

FIG. 10 depicts the nucleic acid sequence for the Fc region of humanFcγ1(YN>AQ)-IL-7 (SEQ ID NO:21).

FIG. 11 is the nucleic acid sequence for the Fc region of humanFcγ2(h)-IL-7 (SEQ ID NO:20).

FIG. 12 is the nucleic acid sequence for the Fc region of human Fcγ2(h)(FN>AQ)-IL-7 (SEQ ID NO:19)

FIG. 13 is a graphical representation of the pharmacokinetic profile ofrecombinant human IL-7 (open squares) and the fusion proteinFcγ2(h)(FN>AQ)-IL-7 (open diamonds) of Example 7. The serumconcentration of the administered IL-7 fusion proteins (in ng/ml) wasmeasured over time (in hours).

FIG. 14 is a graphical representation of B-cell reconstitution inirradiated, bone marrow transplanted mice treated with recombinant humanIL-7 (open symbols), human Fc-IL-7 (filled symbols) or PBS (X). Proteinswere administered every other day (squares) or once a week (triangles).The stippled line represents B-cell concentration in donor mice.

FIG. 15 is a graphical representation of T-cell reconstitution inirradiated, bone marrow transplanted mice treated with recombinant humanIL-7 (open symbols), human Fc-IL-7 (filled symbols) or PBS (X). Proteinswere administered every other day (squares) or once a week (triangles).The stippled line represents T-cell concentration in donor mice.

FIG. 16 is a dot plot representing lymphocyte populations of samplesfrom the blood (top row) and spleen (bottom row) of irradiated, bonemarrow transplanted mice treated with huFcγ2(h)(FN>AQ)-IL-7 (first twocolumns), and untreated controls (last column). The first columnrepresents reconstituted endogenous lymphocytes (CD45.2+), and thesecond column represents reconstituted donor lymphocytes (CD45.1+).T-Lymphocytes were detected as CD3 positive cells shown in the lowerright quadrant. B-lymphocytes were detected as B220 positive cells,shown in the upper left quadrant.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides IL-7 fusion proteins that have enhancedbiological activity compared to wild-type IL-7 proteins. In particular,the invention provides IL-7 fusion proteins that include animmunoglobulin (Ig) portion. These Ig-IL-7 fusion proteins have enhancedbiological activity, such as extended serum half-life as compared to thewild type IL-7 proteins, which makes them suitable for use in thetreatment of conditions accompanied by immune cell deficiencies such aslymphocyte deficiencies.

The invention is further based in part on the finding that IL-7 fusionproteins that have particular structural characteristics also haveenhanced biological properties. While the amino acid sequence ofmammalian IL-7 is well known, information about the structure ofeukaryotically derived IL-7 proteins, including, for example, how theprotein folds and the effects of its predicted N-linked glycosylationsites on its biological activity, remain ill-defined. For example, humanIL-7 protein has a cysteine at positions 2, 34, 47, 92, 129, and 141 ofthe mature protein and three potential N-linked glycosylation sites atpositions asparagine (Asn)70, Asn91, and Asn116. However, the precisestructure of IL-7 synthesized under eukaryotic conditions is unknown.

The present invention includes IL-7 fusion proteins having particularstructural forms and enhanced biological activity. For example, IL-7fusion proteins having the disulfide bonding pattern of Cys2-Cys92,Cys34-Cys129 and Cys47-141 are more active in vivo than a wild-typerecombinant IL-7 protein.

Moreover, the invention provides a form of an IL-7 fusion protein inwhich only two of the three potential N-linked glycosylation sites ofIL-7 are glycosylated. Specifically, Asn70 and Asn91 of the matureprotein are glycosylated, while the predicted N-linked glycosylationsite at IL-7 Asn116 is not glycosylated. Such an IL-7 fusion protein ismore active in vivo than a wild-type recombinant IL-7.

The invention also includes IL-7 fusion proteins wherein the IL-7 moietycontains a deletion and which retain comparable activity compared to thecorresponding unmodified IL-7 fusion proteins. For example, theinvention provides a form of Ig-IL-7 in which the IL-7 moiety containsan eighteen amino acid internal deletion corresponding to the sequenceVKGRKPAALGEAQPTKSL (SEQ ID NO:9).

Interleukin-7 Fusion Proteins

Typically, the IL-7 protein portion is fused to a carrier protein. Inone embodiment, the carrier protein is disposed towards the N-terminusof the fusion protein and the IL-7 protein is disposed towards theC-terminus. In another embodiment, the IL-7 fusion protein is disposedtowards the N-terminus of the fusion protein and the carrier protein isdisposed towards the C-terminus.

As used herein, the term “interleukin-7” or “IL-7” mean IL-7polypeptides and derivatives and analogs thereof having substantialamino acid sequence identity to wild-type mature mammalian IL-7s andsubstantially equivalent biological activity, e.g., in standardbioassays or assays of IL-7 receptor binding affinity. For example, IL-7refers to an amino acid sequence of a recombinant or non-recombinantpolypeptide having an amino acid sequence of: i) a native ornaturally-occurring allelic variant of an IL-7 polypeptide, ii) abiologically active fragment of an IL-7 polypeptide, iii) a biologicallyactive polypeptide analog of an IL-7 polypeptide, or iv) a biologicallyactive variant of an IL-7 polypeptide. IL-7 polypeptides of theinvention can be obtained from any species, e.g., human, cow or sheep.IL-7 nucleic acid and amino acid sequences are well known in the art.For example, the human IL-7 amino acid sequence has a Genbank accessionnumber of NM 000880 (SEQ ID NO:1) and is shown in FIG. 1; the mouse IL-7amino acid sequence has a Genbank accession number of NM 008371; the ratIL-7 amino acid sequence has a Genbank accession number of AF 367210;the cow IL-7 amino acid sequence has a Genbank accession number of NM173924 (SEQ ID NO:2) and is shown in FIG. 2; and the sheep IL-7 aminoacid sequence has a Genbank accession number of U10089 (SEQ ID NO:3) andis shown in FIG. 3. The signal sequence for each of the polypeptidespecies is shown in bold in each of the figures and is typically notincluded where the IL-7 portion is fused C-terminal to the carrierprotein.

A “variant” of an IL-7 protein is defined as an amino acid sequence thatis altered by one or more amino acids. The variant can have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant can have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations can also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological activitycan be found using computer programs well known in the art, for examplesoftware for molecular modeling or for producing alignments. The variantIL-7 proteins included within the invention include IL-7 proteins thatretain IL-7 activity. IL-7 polypeptides which also include additions,substitutions or deletions are also included within the invention aslong as the proteins retain substantially equivalent biological IL-7activity. For example, truncations of IL-7 which retain comparablebiological activity as the full length form of the IL-7 protein areincluded within the invention. The activity of the IL-7 protein can bemeasured using in vitro cellular proliferation assays such as describedin Example 6 below. The activity of IL-7 variants of the inventionmaintain biological activity of at least 10%, 20%, 40%, 60%, but morepreferably 80%, 90%, 95% and even more preferably 99% as compared towild type IL-7.

Variant IL-7 proteins also include polypeptides that have at least about70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity with wild-type IL-7. To determine the percent identity of twoamino acid sequences or of two nucleic acids, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in thesequence of a first amino acid or nucleic acid sequence for optimalalignment with a second amino acid or nucleic acid sequence). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions.times.100). The determinationof percent homology between two sequences can be accomplished using amathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

Potential T-cell of B-cell epitopes in the IL-7 moiety can be removed ormodified in the Fc-IL-7 fusion proteins of the invention. Exemplarydeimmunized IL-7 moieties are disclosed in U.S. Provisional PatentApplication No. 60/634,470.

Carrier Protein

The carrier protein can be any moiety covalently fused to the IL-7protein. In one embodiment, the carrier protein is albumin, for example,human serum albumin. In another embodiment, the carrier protein is animmunoglobulin (Ig) moiety, such as an Ig heavy chain. The Ig chain maybe derived from IgA, IgD, IgE, IgG, or IgM. According to the invention,the Ig moiety may form an intact antibody and may direct the IL-7 fusionprotein to specific target sites in the body. Fusion proteins making useof antibody targeting are known to those in the art. In anotherembodiment, the carrier Ig moiety further comprises an Ig light chain.

In one embodiment, the Ig moiety comprises an Fc region. As used herein,“Fc portion” encompasses domains derived from the constant region of animmunoglobulin, preferably a human immunoglobulin, including a fragment,analog, variant, mutant or derivative of the constant region. Suitableimmunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes. Theconstant region of an immunoglobulin is defined as a naturally-occurringor synthetically-produced polypeptide homologous to the immunoglobulinC-terminal region, and can include a CH1 domain, a hinge, a CH2 domain,a CH3 domain, or a CH4 domain, separately or in any combination. In thepresent invention, the Fc portion typically includes at least a CH2domain. For example, the Fc portion can include hinge-CH2—CH3.Alternatively, the Fc portion can include all or a portion of the hingeregion, the CH2 domain and/or the CH3 domain and/or the CH4 domain.

The constant region of an immunoglobulin is responsible for manyimportant antibody functions including Fc receptor (FcR) binding andcomplement fixation. There are five major classes of heavy chainconstant region, classified as IgA, IgG, IgD, IgE, and IgM. For example,IgG is separated into four γ subclasses: γ1, γ2, γ3, and γ4, also knownas IgG1, IgG2, IgG3, and IgG4, respectively.

IgG molecules interact with multiple classes of cellular receptorsincluding three classes of Fcγreceptors (FcγR) specific for the IgGclass of antibody, namely FcγRI, FcγRII, and FcγRIII. The importantsequences for the binding of IgG to the FcγR receptors have beenreported to be located in the CH2 and CH3 domains. The serum half-lifeof an antibody is influenced by the ability of that antibody to bind toan Fc receptor (FcR). Similarly, the serum half-life of immunoglobulinfusion proteins is also influenced by the ability to bind to suchreceptors (Gillies et al., (1999) Cancer Res. 59:2159-66). Compared tothose of IgG1, CH2 and CH3 domains of IgG2 and IgG4 have biochemicallyundetectable or reduced binding affinity to Fc receptors. It has beenreported that immunoglobulin fusion proteins containing CH2 and CH3domains of IgG2 or IgG4 had longer serum half-lives compared to thecorresponding fusion proteins containing CH2 and CH3 domains of IgG1(U.S. Pat. No. 5,541,087; Lo et al., (1998) Protein Engineering,11:495-500). Accordingly, in certain embodiments of the invention,preferred CH2 and CH3 domains are derived from an antibody isotype withreduced receptor binding affinity and effector functions, such as, forexample, IgG2 or IgG4. More preferred CH2 and CH3 domains are derivedfrom IgG2.

The hinge region is normally located C-terminal to the CH1 domain of theheavy chain constant region. In the IgG isotypes, disulfide bondstypically occur within this hinge region, permitting the finaltetrameric antibody molecule to form. This region is dominated byprolines, serines and threonines. When included in the presentinvention, the hinge region is typically at least homologous to thenaturally-occurring immunoglobulin region that includes the cysteineresidues to form disulfide bonds linking the two Fc moieties.Representative sequences of hinge regions for human and mouseimmunoglobulins are known in the art and can be found in Borrebaeck,ed., (1992) Antibody Engineering, A Practical Guide, W. H. Freeman andCo. Suitable hinge regions for the present invention can be derived fromIgG1, IgG2, IgG3, IgG4, and other immunoglobulin classes.

The IgG1 hinge region has three cysteines, the second and third of whichare involved in disulfide bonds between the two heavy chains of theimmunoglobulin. These same two cysteines permit efficient and consistentdisulfide bonding of an Fc portion. Therefore, a preferred hinge regionof the present invention is derived from IgG1, more preferably fromhuman IgG1, wherein the first cysteine is preferably mutated to anotheramino acid, preferably serine.

The IgG2 isotype hinge region has four disulfide bonds that tend topromote oligomerization and possibly incorrect disulfide bonding duringsecretion in recombinant systems. A suitable hinge region can be derivedfrom an IgG2 hinge; the first two cysteines are each preferably mutatedto another amino acid.

The hinge region of IgG4 is known to form interchain disulfide bondsinefficiently. However, a suitable hinge region for the presentinvention can be derived from the IgG4 hinge region, preferablycontaining a mutation that enhances correct formation of disulfide bondsbetween heavy chain-derived moieties (Angal et al. (1993) Mol. Immunol.,30:105-8).

In accordance with the present invention, the Fc portion can contain CH2and/or CH3 and/or CH4 domains and a hinge region that are derived fromdifferent antibody isotypes, i.e., a hybrid Fc portion. For example, inone embodiment, the Fc portion contains CH2 and/or CH3 domains derivedfrom IgG2 or IgG4 and a mutant hinge region derived from IgG1. As usedin this application, Fcγ2(h) refers to an embodiment wherein the hingeis derived from IgG1 and the remaining constant domains are from IgG2.Alternatively, a mutant hinge region from another IgG subclass is usedin a hybrid Fc portion. For example, a mutant form of the IgG4 hingethat allows efficient disulfide bonding between the two heavy chains canbe used. A mutant hinge can also be derived from an IgG2 hinge in whichthe first two cysteines are each mutated to another amino acid. Suchhybrid Fc portions facilitate high-level expression and improve thecorrect assembly of the Fc-IL-7 fusion proteins. Assembly of such hybridFc portions is known in the art and has been described in U.S. PublishedPatent Application No. 2003-0044423.

In some embodiments, the Fc portion contains amino acid modificationsthat generally extend the serum half-life of an Fc fusion protein. Suchamino acid modifications include mutations substantially decreasing oreliminating Fc receptor binding or complement fixing activity. Forexample, the glycosylation site within the Fc portion of animmunoglobulin heavy chain can be removed. In IgG1, the glycosylationsite is Asn297 within the amino acid sequence Gln-Tyr-Asn-Ser (SEQ IDNO:30). In other immunoglobulin isotypes, the glycosylation sitecorresponds to Asn297 of IgG1. For example, in IgG2 and IgG4, theglycosylation site is the asparagine within the amino acid sequenceGln-Phe-Asn-Ser (SEQ ID NO:29). Accordingly, a mutation of Asn297 ofIgG1 removes the glycosylation site in an Fc portion derived from IgG1.In one embodiment, Asn297 is replaced with Gln. In other embodiments,the tyrosine within the amino acid sequence Gln-Tyr-Asn-Ser (SEQ IDNO:30) is further mutated to eliminate a potential non-self T-cellepitope resulting from asparagine mutation. As used herein, a T-cellepitope is a polypeptide sequence in a protein that interacts with orbinds an MHC class II molecule. For example, the amino acid sequenceGln-Tyr-Asn-Ser (SEQ ID NO:30) within an IgG1 heavy chain can bereplaced with a Gln-Ala-Gln-Ser (SEQ ID NO:28) amino acid sequence.Similarly, in IgG2 or IgG4, a mutation of asparagine within the aminoacid sequence Gln-Phe-Asn-Ser (SEQ ID NO:29) removes the glycosylationsite in an Fc portion derived from IgG2 or IgG4 heavy chain. In oneembodiment, the asparagine is replaced with a glutamine. In otherembodiments, the phenylalanine within the amino acid sequenceGln-Phe-Asn-Ser (SEQ ID NO:29) is further mutated to eliminate apotential non-self T-cell epitope resulting from asparagine mutation.For example, the amino acid sequence Gln-Phe-Asn-Ser (SEQ ID NO:29)within an IgG2 or IgG4 heavy chain can be replaced with aGln-Ala-Gln-Ser (SEQ ID NO:28) amino acid sequence.

It has also been observed that alteration of amino acids near thejunction of the Fc portion and the non-Fc portion can dramaticallyincrease the serum half-life of the Fc fusion protein. (U.S. PublishedPatent Application No. 2002-0147311). Accordingly, the junction regionof an Fc-IL-7 or IL-7-Fc fusion protein of the present invention cancontain alterations that, relative to the naturally-occurring sequencesof an immunoglobulin heavy chain and IL-7, preferably lie within about10 amino acids of the junction point. These amino acid changes can causean increase in hydrophobicity by, for example, changing the C-terminallysine of the Fc portion to a hydrophobic amino acid such as alanine orleucine. In yet another embodiment of the invention, the C-terminallysine and preceding glycine of the Fc portion is deleted.

In other embodiments, the Fc portion contains amino acid alterations ofthe Leu-Ser-Leu-Ser segment near the C-terminus of the Fc portion of animmunoglobulin heavy chain. The amino acid substitutions of theLeu-Ser-Leu-Ser (SEQ ID NO:27) segment eliminate potential junctionalT-cell epitopes. In one embodiment, the Leu-Ser-Leu-Ser (SEQ ID NO:27)amino acid sequence near the C-terminus of the Fc portion is replacedwith an Ala-Thr-Ala-Thr (SEQ ID NO:26) amino acid sequence. In otherembodiments, the amino acids within the Leu-Ser-Leu-Ser (SEQ ID NO:27)segment are replaced with other amino acids such as glycine or proline.Detailed methods of generating amino acid substitutions of theLeu-Ser-Leu-Ser (SEQ ID NO:27) segment near the C-terminus of an IgG1,IgG2, IgG3, IgG4, or other immunoglobulin class molecules, as well asother exemplary modifications for altering junctional T-cell epitopes,have been described in U.S. Published Patent Application No.2003-0166877.

Spacer

In one embodiment, a spacer or linker peptide is inserted between thecarrier protein and the IL-7 fusion protein. For example, the spacer isplaced immediately C-terminal to the last amino acid of an Ig constantregion. The spacer or linker peptide is preferably non-charged and morepreferably non-polar or hydrophobic. The length of a spacer or linkerpeptide is preferably between 1 and about 100 amino acids, morepreferably between 1 and about 50 amino acids, or between 1 and about 25amino acids, and even more preferably between 1 and about 15 aminoacids, and even more preferably less than 10 amino acids. In oneembodiment, the spacer contains a sequence (G₄S)_(n), where n is lessthan 5. In a preferred embodiment, the spacer contains the sequenceG₄SG₄ (SEQ ID NO:25). In yet another embodiment, the spacer contains amotif that is recognized as an N-linked glycosylation site. In yetanother embodiment, the spacer contains a motif that is recognized by asite specific cleavage agent. In an alternative embodiment of theinvention, the carrier protein and IL-7 fusion protein are separated bya synthetic spacer, for example a PNA spacer, that is preferablynon-charged, and more preferably non-polar or hydrophobic.

Production of IL-7 Fusion Proteins

Non-limiting methods for synthesizing useful embodiments of theinvention are described in the Examples herein, as well as assays usefulfor testing the in vitro properties, and pharmacokinetic and in vivoactivities in animal models.

The IL-7 fusion proteins of the invention can be produced usingrecombinant expression vectors known in the art. The term “expressionvector” refers to a replicable DNA construct used to express DNA whichencodes the desired IL-7 fusion protein and which includes atranscriptional unit comprising an assembly of (1) genetic element(s)having a regulatory role in gene expression, for example, promoters,operators, or enhancers, operatively linked to (2) a DNA sequenceencoding the desired IL-7 fusion protein which is transcribed into mRNAand translated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences. The choice of promoterand other regulatory elements generally varies according to the intendedhost cell. A preferred expression vector of the invention is an Fcexpression vector derived from the PdCs-huFc expression vector describedin Lo et al., Protein Engineering (1998) 11:495.

In a preferred example, the nucleic acid encoding the IL-7 fusionprotein is transfected into a host cell using recombinant DNAtechniques. In the context of the present invention, the foreign DNAincludes a sequence encoding the inventive proteins. Suitable host cellsinclude prokaryotic, yeast or higher eukaryotic cells. Preferred hostcells are eukaryotic cells.

The recombinant IL-7 fusion proteins can be expressed in yeast hosts,preferably from Saccharomyces species, such as S. cerevisiae. Yeast ofother genera such as Pichia or Kluyveromyces may also be employed. Yeastvectors will generally contain an origin of replication from a yeastplasmid or an autonomously replicating sequence (ARS), a promoter, DNAencoding the IL-7 fusion protein, sequences for polyadenylation andtranscription termination and a selection gene. Suitable promotersequences in yeast vectors include the promoters for metallothionein,3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-4-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase and glucokinase.

Various mammalian or insect cell culture systems can be employed toexpress recombinant protein. Baculovirus systems for production ofproteins in insect cells are well known in the art. Examples of suitablemammalian host cell lines include NS/0 cells, L cells, C127, 3T3,Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Additionalsuitable mammalian host cells include CV-1 cells (ATCC CCL70) and COS-7cells both derived from monkey kidney. Another suitable monkey kidneycell line, CV-1/EBNA, was derived by transfection of the CV-1 cell linewith a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) andwith a vector containing CMV regulatory sequences (McMahan et al.,(1991) EMBO J. 10:2821). The EBNA-1 gene allows for episomal replicationof expression vectors, such as HAV-EO or pDC406, that contain the EBVorigin of replication.

Mammalian expression vectors may comprise non-transcribed elements suchas an origin of replication, a suitable promoter and enhancer linked tothe gene to be expressed, and other 5′ or 3′ flanking nontranscribedsequences, and 5′ or 3′ nontranslated sequences, such as necessaryribosome binding sites, a poly-adenylation site, splice donor andacceptor sites, and transcriptional termination sequences. Commonly usedpromoters and enhancers are derived from Polyoma, Adenovirus 2, SimianVirus 40 (SV40), and human cytomegalovirus. DNA sequences derived fromthe SV40 viral genome, for example, SV40 origin, early and latepromoter, enhancer, splice, and polyadenylation sites may be used toprovide the other genetic elements required for expression of aheterologous DNA sequence.

For secretion of the IL-7 fusion protein from the host cell, theexpression vector comprises DNA encoding a signal or leader peptide. Inthe present invention, DNA encoding the native signal sequence of IL-7can be used, or alternatively, a DNA encoding a heterologous signalsequence may be used, such as the signal sequence from anotherinterleukin or from a secreted Ig molecule.

The present invention also provides a process for preparing therecombinant proteins of the present invention including culturing a hostcell transformed with an expression vector comprising a DNA sequencethat encodes the IL-7 fusion protein under conditions that promoteexpression. The desired protein is then purified from culture media orcell extracts. For example, supernatants from expression systems thatsecrete recombinant protein into the culture medium can be firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a suitable purification matrix, as known in the art. For example,Fc-IL-7 fusion proteins are conveniently captured using a matrix coupledto Protein A.

An “isolated” or “purified” IL-7 fusion protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theIL-7 fusion protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of IL-7fusion protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of IL-7 fusion protein havingless than about 30% (by dry weight) of non-IL-7 fusion protein (alsoreferred to herein as a “contaminating protein”), more preferably lessthan about 20% of non-IL-7 fusion protein, still more preferably lessthan about 10% of non-IL-7 fusion protein, and most preferably less thanabout 5% non-IL-7 fusion protein. When the IL-7 fusion protein orbiologically active portion thereof is purified from a recombinantsource, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

The term “substantially pure Ig-IL-7 fusion protein” refers to apreparation in which the Ig-IL-7 fusion protein constitutes at least60%, 70%, 80%, 90%, 95% or 99% of the proteins in the preparation. Inone embodiment, the invention includes substantially pure preparationsof Ig-IL-7 fusion proteins having a disulfide bonding pattern betweenCys2 and Cys92, Cys34 and Cys129, and Cys47 and Cys141. In anotherembodiment, the invention features substantially pure preparations ofIg-IL-7 fusion proteins where Asn116 is non-glycosylated but Asn70 andAsn91 are glycosylated.

Methods of Treatment Using Fc-IL-7 Proteins

The IL-7 fusion proteins of the invention are useful in treating immunedeficiencies and in accelerating the natural reconstitution of theimmune system that occurs, for example, after diseases or treatmentsthat are immunosuppressive in nature. For example, IL-7 fusion proteinscan be used to treat infectious pathogens, immune disorders, and toenhance the growth (including proliferation) of specific immune celltypes. Moreover, the IL-7 fusion proteins can be used in the treatmentof cancers such as bladder cancer, lung cancer, brain cancer, breastcancer, skin cancer, and prostate cancer. In one example, it is usefulto treat patients who have undergone one or more cycles of chemotherapywith IL-7 fusion proteins as described above to help their immune cellsreplenish. Alternatively, IL-7 fusion proteins are useful in adoptiveT-cell transplantations. For example, IL-7 fusion proteins may beadministered to facilitate the expansion and survival of transplantedT-cells, or to expand isolated T-cell populations ex vivo.Alternatively, it is also useful to administer the IL-7 fusion proteinsdescribed above to patients with HIV, the elderly, patients receiving atransplant or other patients with suppressed immune system function.

Administration

The IL-7 fusion proteins of the invention can be incorporated into apharmaceutical composition suitable for administration. Suchcompositions typically comprise the IL-7 fusion protein and apharmaceutically-acceptable carrier. As used herein the language“pharmaceutically-acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Medicaments that contain the IL-7 fusion proteins of the invention canhave a concentration of 0.01 to 100% (w/w), though the amount variesaccording to the dosage form of the medicaments.

Administration dose depends on the body weight of the patients, theseriousness of the disease, and the doctor's opinion. However, it isgenerally advisable to administer about 0.01 to about 10 mg/kg bodyweight a day, preferably about 0.02 to about 2 mg/kg, and morepreferably about 0.5 mg/kg in case of injection. The dose can beadministered once or several times daily according to the seriousness ofthe disease and the doctor's opinion.

Compositions of the invention are useful when co-administered with oneor more other therapeutic agents, for example, a molecule also known tobe useful to replenish blood cells. For example, the molecule may beerythropoietin which is known to be used to replenish red blood cells,G-CSF which is used to replenish neutrophils or GM-CSF which is used toreplenish granulocytes and macrophages.

EXAMPLE 1 Cloning of Human (hu) Fc-IL-7 and huFc-IL-7 Variants

The nucleic acid encoding the mature form of human IL-7 (i.e. lackingits N-terminal signal sequence) is amplified by Polymerase ChainReaction (PCR), using forward and reverse primers that incorporated therestriction sites for Sma I and Xho I, respectively. The amplified PCRproduct is cloned into a pCRII vector (Invitrogen, Carlsbad, Calif.),and its sequence verified. The amino acid sequence of mature IL-7 isshown as SEQ ID NO:1. The Sma I/Xho I digested IL-7 fragment istransferred into a likewise treated pdCs-huFc derived expression vector,resulting in a chimeric sequence between huFc and IL-7, with IL-7 placedin-frame, directly downstream of the sequence encoding a CH3 moiety ofFc (see Lo et al., Protein Engineering (1998) 11:495).

A series of expression vectors are derived from the pdCs-huFc vectorwhich encodes an Fc fragment that generally includes a hinge, a CH2domain, and a CH3 domain of Ig, and which have been engineered toincorporate specific alterations in the Fc region. Thus, by shuttlingthe IL-7 fragment between these vectors, a series of huFc-IL-7 fusionproteins were generated which differ in their Fc backbone. In order tocreate the various backbones, the appropriate mutations can first beintroduced into the Fc sequence by methods known in the art. Since theFc region of the pdCs-huFc derived vector is flanked by an AflIIrestriction site and a SmaI restriction site, by subjecting the nucleicacid of the appropriately modified backbone to PCR using primers thatincorporate the restriction sites for AflII and SmaI respectively, theresultant Fc encoding nucleic acid fragment can then be substituted intothe pdCs-huFc derived vector as an AflII to SmaI fragment. The AflIIsequence CTTAAGC (SEQ ID NO:24) is upstream of the Fc sequence beginningGAGCCCAAA (SEQ ID NO:23), which represents the beginning of the hingeregion as shown in FIG. 20. The SmaI site CCCGGGT (SEQ ID NO:17) istowards the end of the CH3 region as shown by the underlined nucleicacids in FIG. 12 and encodes the Pro-Gly amino acids proceeding thealanine residue from the lysine to alanine mutation at the end of theCH3 region.

For example, huFcγ1-IL-7 is constructed having the hinge region, the CH2and the CH3 domains derived from the IgGγ1 subclass. In the context ofan Fc fusion protein, the IgGγ1 hinge region in addition contains amutation substituting the first cysteine for serine. The sequence of theencoded fusion protein is depicted in FIG. 4 (SEQ ID NO:4), while thesequence in SEQ ID NO:22 encodes the mature huFcγ1 backbone of thevector.

In addition, Fcγ1-IL-7 fusion proteins were generated that included thedipeptide mutation YN to AQ to eliminate the glycosylation site on Fc(corresponding to N297 in IgGγ1) as well as a potential immunogenicT-cell epitope, according to the methods described above. The mature Fcbackbone sequence for huFcγ1(YN>AQ) is disclosed in SEQ ID NO:21. Thesubstitution of alanine and glycine in place of tyrosine and asparaginewas accomplished by first introducing mutations into the Fc backbone byan overlap PCR approach. Two overlapping complementary mutagenic primerswere used to generate two PCR fragments, which were used as the templatein a second round of amplification to produce a single fragmentcontaining the appropriate codon substitutions. The mutagenic primer inthe sense direction was 5′-AGCAGGCCCAGAG CACGTACCGTGTGGT-3′ (mutationunderlined) (SEQ ID NO:36). The complementary strand was5′-GTACGTGCTCTGGGCCTGCTCCTCCCGC-3′ (SEQ ID NO:37). The flanking forwardprimer was 5′-CTCTCTGCAGAGCCCAAA TCT-3′ (SEQ ID NO:38), which alsocontains a PstI site. In the antisense direction, the flanking reverseprimer was 5′CAGGGTGTACACCTGTGGTTC-3′ (SEQ ID NO:33), which alsocontains a BsrGI site. After amplification, the sequence was verifiedthrough standard methods and was subject to restriction by BsrGI andPstI. The resultant fragment was then substituted for the non-mutantfragment of the Fc region.

The huFcγ2(h)(FN>AQ)-IL-7 was also constructed using the techniquespreviously described. This fusion protein includes an altered hingeregion which was derived from IgGγ1 subclass, while the CH2 and CH3domains were derived from IgGγ2 subclass. Additionally, the dipeptidemutation FN to AQ was included to eliminate the glycosylation site on Fc(corresponding to N297 in IgGγ1) as well as a potential immunogenicT-cell epitope. The sequence of the encoded fusion protein is depictedin FIG. 5 (SEQ ID NO:5). The sequence of the mature Fc backbonehuFcγ2(h)(FN>AQ) is shown in SEQ ID NO:19).

In addition, Fc-IL-7 fusion proteins were generated which included aflexible linker sequence between the Fc moiety and the IL-7 moiety. Forexample, a linker polypeptide with the sequence GGGGSGGGGSGGGGS(linker1, SEQ ID NO:34) was inserted. To generate huFcγ1(linker1)-IL-7,a synthetic oligonucleotide duplex of the sequence 5′-G GGT GCA GGG GGCGGG GGC AGC GGG GGC GGA GGA TCC GGC GGG GGC TC-3′ (SEQ ID NO:18) wasinserted by blunt-end ligation at the unique SmaI site of the expressionvector pdCs-huFc-IL-7 and the orientation of the duplex was verified.The forward primer was designed such that the amino acid residuesPro-Gly encoded by the codons spanning the SmaI site (C CCG GGT) (SEQ IDNO:17), and the ensuing Ala residue (resulting from the encoded lysineto alanine substitution) of the CH3 region, were maintained. The aminoacid sequence of the encoded fusion protein is shown in FIG. 6 (SEQ IDNO:6).

Additional Fc-IL-7 fusion proteins were constructed that included ashorter linker polypeptide with the sequence GGGGSGGGG (linker2, SEQ IDNO:25). To generate huFcγ1(YN>AQ)(linker2)-IL-7, an amplified PCRproduct, obtained from the primer pair5′-CCCGGGCGCCGGCGGTGGAGGATCAGGTGGTGGCGGTGAT TGTGA TATTGAAGGTAAAGATG-3′(containing the encoded linker sequence, SEQ ID NO:15) and5′-ATCATGTCTGGATCCCTCGA-3′ (SEQ ID NO:14) on an appropriate pdCs-Fc-IL-7template plasmid, was cloned into a pCRII vector (Invitrogen, Carlsbad,Calif.), and its sequence verified. A Xma I/Xho I digested fragmentencoding linker2/IL-7 was then transferred into a likewise treatedpdCs-huFc derived expression vector. The vector was modified to containthe mature Fc backbone huFcγ1(YN>AQ) of SEQ ID NO:21. The amino acidsequence of the encoded fusion protein is shown in FIG. 7 (SEQ ID NO:7).

Similarly, huFcγ1(YN>AQ,d)(linker2)-IL-7 was generated, using the primerpair 5′-CCCGGGCGGTGGAGGATCAGGTGGTGGCGGTGATTGTGATAT TGAAGGTAAAGATG-3′(SEQ ID No:16) and 5′-ATCATGTCTGGATCCCTCGA-3′ (SEQ ID NO:12).huFcγ1(YN>AQ,d)(linker2)-IL-7 differs from the preceding fusion proteinhuFcγ1(YN>AQ)(linker2)-IL-7 in that it lacks the terminal two amino acidresidues of the Fc portion of the fusion protein. Specifically, ratherthan terminating in with the sequence . . . ATATPGA (SEQ ID NO:11), theFc portion ends with the sequence . . . ATATP (SEQ ID NO:10). The aminoacid sequence of the encoded fusion protein is shown in FIG. 8 (SEQ IDNO:8).

EXAMPLE 2 Transfection and Expression of Fc-IL-7 Fusion Proteins

Electroporation was used to introduce the DNA encoding the IL-7 fusionproteins described above into a mouse myeloma NS/0 cell line. To performelectroporation NS/0 cells were grown in Dulbecco's modified Eagle'smedium supplemented with 10% heat-inactivated fetal bovine serum, 2 mMglutamine and penicillin/streptomycin. About 5×10⁶ cells were washedonce with PBS and resuspended in 0.5 ml PBS. 10 μg of linearized plasmidDNA for huFcγ1-IL-7 was then incubated with the cells in a Gene PulserCuvette (0.4 cm electrode gap, BioRad) on ice for 10 min.Electroporation was performed using a Gene Pulser (BioRad, Hercules,Calif.) with settings at 0.25 V and 500 μF. Cells were allowed torecover for 10 min on ice, after which they were resuspended in growthmedium and plated onto two 96 well plates.

Stably transfected clones were selected by their growth in the presenceof 100 nM methotrexate (MTX), which was added to the growth medium twodays post-transfection. The cells were fed every 3 days for two to threemore times, and MTX-resistant clones appeared in 2 to 3 weeks.Supernatants from clones were assayed by anti-Fc ELISA to identifyclones that produced high amounts of the IL-7 fusion proteins. Highproducing clones were isolated and propagated in growth mediumcontaining 100 nM MTX. Typically, a serum-free growth medium, such asH-SFM or CD medium (Life Technologies), was used.

EXAMPLE 3 Biochemical Analysis of huFc-IL-7 Fusion Proteins

Routine SDS-PAGE characterization was used to assess the integrity ofthe fusion proteins. Differences between the huFc-IL-7 variantshuFcγ1-IL-7, huFcγ2(h)(FN>AQ)-IL-7, huFcγ1 (linker1)-IL-7,huFcγ1(YN>AQ)(linker2)-IL-7 and huFcγ1(YN>AQ,d)-IL-7 were investigated.The huFc-IL-7 fusion proteins, expressed from NS/0 cells, were capturedon Protein A Sepharose beads (Repligen, Needham, Mass.) from the tissueculture medium into which they were secreted, and were eluted by boilingin protein sample buffer, with or without a reducing agent such asβ-mercaptoethanol. The samples were separated by SDS-PAGE and theprotein bands were visualized by Coomassie staining. By SDS-PAGE thetested huFc-IL-7 fusion proteins were generally well expressed, as theywere present substantially as a single band on the gel; it was foundthat in samples of huFc-IL-7 variants that included a linker, secondarybands, which may represent clipped material, were noticeably reduced.

Purified huFc-IL-7 fusion proteins were also analyzed by size exclusionchromatography (SEC) to assess the extent to which the huFc-IL-7variants were aggregated. Briefly, the cell culture supernatant wasloaded onto a pre-equilibrated Fast-Flow Protein A Sepharose column, thecolumn was washed extensively in a physiological buffer (such as 100 mMSodium Phosphate, 150 mM NaCl at neutral pH), and the bound protein waseluted at about pH 2.5 to 3 in same salt buffer as above. Fractions wereimmediately neutralized.

It was found that for each of the fusion proteins tested at least 50% ofthe product was monomeric, and generally more than 65%. “Monomeric,” asused herein, refers to non-aggregated proteins. It is understood thatproteins with an Fc portion normally form a disulfide-bonded complexwhich normally include two polypeptide chains (unless the two Fcportions are present within the same polypeptide) and may be thought ofas a “unit-dimer”. “Monomeric” is not intended to exclude suchdisulfide-bonded species, but only to connote that the proteins arenon-aggregated. To obtain a virtually monomeric huFc-IL-7 fusion proteinpreparation (around 98%), the eluate from a Sepharose-ProteinApurification was loaded onto a preparative SEC column (Superdex) and themonomeric peak fraction was collected. Typically, the concentration ofthe recovered protein was around 1 mg/ml. If required, the sample wasconcentrated, for example by spin dialysis (e.g. VivaSpin) with amolecular weight cut-off of 10-30 kDa.

Disulfide Bonding

IL-7 contains six Cys residues which could make disulfide bonds, atpositions Cys2, Cys34, Cys47, Cys92, Cys129 and Cys141 of the matureIL-7 protein sequence. The folding of huFcγ1-IL-7 was assessed bydetermining the pattern of disulfide bonds present in the IL-7 moiety ofthe fusion protein. Briefly, peptide maps of huFcγ1-IL-7 were generatedfrom trypsinized material and analyzed for the presence of signaturepeptide fragments. huFcγ1-IL-7 protein was trypsinized either in anative form or after reduction and alkylation. To account for peptidefragments that may be glycosylated, samples of the native and denaturedproteins were additionally treated with PNGaseF to remove glycosylchains prior to tryptic digestion. Peptide fragments were fractionatedby HPLC, and their mass was determined by mass spectroscopy.

In the context of Fcγ1-IL-7, a peptide fragment containing the disulfidebond Cys47-Cys141 (“3-6”) would be predicted to have a mass of 1447.6,whereas a peptide fragment containing the disulfide bond Cys2-Cys141(“1-6”) would be predicted to have a mass of 1426.6. Similarly, apeptide fragment containing the disulfide bond Cys34-Cys129 (“2-5”)would be predicted to have a mass of 2738.3. Indeed, peptide fragmentsof a mass of 1447.6 (“3-6”) and of 2738.3 (“2-5”) were identified insamples derived from the native Fc-IL-7 protein regardless of whetherthe samples were treated with PNGaseF or not, but not in samples fromreduced Fc-IL-7. Conversely, the peptide fragment of a mass of 1426.6(“1-6”) was not found in any sample. Thus, Fcγ1-IL-7 contained thedisulfide bonds Cys47-Cys141 and Cys34-Cys129, but not Cys2-Cys141. Itwas noted that a peptide fragment of the predicted mass of 2439.2,corresponding to a fragment containing Cys2-Cys92 (“1-4”) was identifiedonly in the sample from the native fusion protein treated with PNGaseF.In fact, Cys92 lies within the tripeptide motif Asn91Cys92Thr93,indicating that Asn91 was glycosylated in huFcγ1-IL-7. Thus, inhuFcγ1-IL-7 the disulfide bonding pattern was consistent withCys2-Cys92, Cys34-Cys129, and Cys47-Cys141. This experimentallydetermined configuration of disulfide bonds of Fc-IL-7 stands incontrast to the experimentally determined configuration reported forbacterially produced and re-folded IL-7 (Cosenza et al. (1997) JBC272:32995).

N-Linked Glycosylation Sites

Human IL-7 contains three potential glycosylation sites, at positionsAsn70, Asn91 and Asn116 of the mature IL-7 protein sequence. Peptidemaps of huFcγ1-IL-7 (reduced/alkylated) were analyzed for the presenceof signature peptide fragments. If glycosylated, these signaturefragments would only be revealed in samples treated with PNGaseF. Massesof 1489.7, 1719.9 and 718.3 would be predicted for tryptic peptidefragments containing the unmodified residues for Asn70, Asn91, andAsn116, respectively.

Indeed, peptide fragments of a mass of 1489.7 and of 1719.9 wereidentified in samples that had been treated with PNGaseF, but wereabsent in the untreated sample, indicating that Asn70 (contained in thesequence . . . MNSTG . . . ) (SEQ ID NO:31), and Asn91 (contained in thesequence . . . LNCTG . . . ) (SEQ ID NO:32), were indeed glycosylated.Surprisingly, a tryptic fragment of a mass of 718.3, corresponding toSLEENK (SEQ ID NO:35), was identified in both the PNGaseF treated sampleand the untreated sample, indicating that Asn116 was not glycosylated.This was not expected because Asn116 in the human IL-7 sequence . . .PTKSLEENKSLKE . . . (SEQ ID NO:13) (see SEQ ID NO:1) is predicted to bean N-linked glycosylation site. The NKS putative glycosylation site isconserved in sheep and cows as well as humans.

The analysis of disulfide bonding patterns and N-linked glycosylationsites, repeated with samples of Fcγ1-(linker1)-IL-7 and ofFcγ2h(FN>AQ)-IL-7, gives similar results.

EXAMPLE 4 ELISA Procedures

The concentrations of protein products in the supernatants ofMTX-resistant clones and other test samples was determined by anti-huFcELISA, as described in detail below. ELISA plates were coated withAffiniPure Goat anti-Human IgG (H+L) (Jackson Immuno ResearchLaboratories, West Grove, Pa.) at 5 μg/mL in PBS, and 100 μL/well in96-well plates. Coated plates were covered and incubated at 4° C.overnight. Plates were washed 4 times with 0.05% Tween (Tween 20) inPBS, and blocked with 1% BSA/1% goat serum in PBS, 200 μL/well. Afterincubation with the blocking buffer at 37° C. for 2 hrs, the plates werewashed 4 times with 0.05% Tween in PBS and tapped dry. Test samples werediluted as appropriate in sample buffer (1% BSA/1% goat serum/0.05%Tween in PBS). A standard curve was prepared using a chimeric antibody(with a human Fc) of known concentration. To prepare a standard curve,serial dilutions were made in the sample buffer to give a standard curveranging from 125 ng/mL to 3.9 ng/mL. The diluted samples and standardswere added to the plate, 100 μL/well and the plate incubated at 37° C.for 2 hr. After incubation, the plate was washed 8 times with 0.05%Tween in PBS. To each well 100 μL of the secondary antibody horseradishperoxidase-conjugated anti-human IgG was added, diluted to around1:120,000 in sample buffer. The exact dilution of the secondary antibodywas determined for each lot of the HRP-conjugated anti-human IgG. Afterincubation at 37° C. for 2 hr, the plate was washed 8 times with 0.05%Tween in PBS.

The substrate solution was added to the plate at 100 μL/well. Thissolution was prepared by dissolving 30 mg of OPD (o-phenylenediaminedihydrochloride (OPD), (1 tablet) into 15 mL of 0.025 M citric acid/0.05M Na₂HPO₄ buffer, pH 5, which contained 0.03% of freshly added hydrogenperoxide. The color was allowed to develop for about 30 minutes at roomtemperature in the dark. The reaction was stopped by adding 4N sulfuricacid, 100 μL/well. The plate was read by a plate reader, which was setat both 490 and 650 nm and programmed to subtract the background OD at650 nm from the OD at 490 nm.

The concentration of human IL-7 in serum samples of animals treated withhuFc-IL-7 fusion proteins or recombinant human IL-7 was determined byELISA, essentially as described above. Human IL-7 was captured via amouse anti-human IL-7 antibody (R&D Systems, Minneapolis, Minn.) anddetected with a goat anti-human IL-7 biotin antibody (R&D Systems,Minneapolis, Minn.).

EXAMPLE 5 Purification of huFc-IL-7 Proteins

A standard purification of Fc-containing fusion proteins was performedbased on the affinity of the Fc protein moiety for Protein A. Briefly,NS/0 cells expressing the appropriate fusion protein, were grown intissue culture medium and the supernatant containing the expressedprotein was collected and loaded onto a pre-equilibrated Past FlowProtein A Sepharose column. The column was then washed extensively withbuffer (such as 100 mM Sodium Phosphate, 150 mM NaCl at neutral pH).Bound protein was eluted at a low pH (pH 2.5-3) in same buffer as aboveand fractions were immediately neutralized.

To obtain a non-aggregated huFc-IL-7 fusion protein preparation (around98% monomer), the eluate was loaded onto a preparative SEC column(Superdex) and the monomeric peak fraction was collected. Typically, theconcentration of the recovered protein was around 0.5 mg/ml to 2 mg/ml,and where appropriate the sample was concentrated by spin dialysis (e.g.Viva Spin with a molecular weight cut-off of 30 kDa).

EXAMPLE 6 In Vitro Activity of huFc-IL-7 Proteins

The cytokine activity of the purified huFc-IL-7 fusion proteins wasdetermined in vitro in a cellular proliferation bioassay. Human PBMC(Peripheral Blood Mononuclear Cells) were activated by PHA-P to producecells which were responsive to IL-7. Proliferation was measured in astandard thymidine incorporation assay. Briefly, PBMC's were firstincubated for five days with 10 microgram/ml PHA-P, cells were washedand then incubated in a medium supplemented with the huFc-IL-7 fusionproteins, in a dilution series, for a total of 48 hours. During thefinal 12 hours, the samples were pulsed with 0.3 μCi of[methyl-3H]thymidine (Dupont-NEN-027). Cells were then washedextensively, harvested and lysed onto glass filters. 3H-thymidineincorporated into DNA was measured in a scintillation counter. As astandard, wild type huIL-7 protein, obtained from R&D Systems(Minneapolis, Minn.), or obtained from the National Institute forBiological Standards and Control (NIBSC), was assayed.

An ED50 value of cell proliferation for huFc-IL-7 fusion proteins wasobtained from plotting a dose response curve according to standardtechniques, and determining the protein concentration that resulted inhalf-maximal response. The fusion proteins huFcγ1-IL-7,huFcγ2(h)(FN>AQ)-IL-7, and huFcγ1(linker1)-IL-7 were evaluated. The ED50values of the fusion proteins were fairly similar to one another,falling within a 3-fold range from one another. Therefore, it was foundthat these alterations in the Fc moiety have little influence on IL-7activity of the fusion protein.

In addition, it was found that the ED50 values of these fusion proteinswere about 3- to 10-fold higher than the ED50 value obtained for huIL-7commercially available from R&D Systems. Since this commercialpreparation is produced in bacteria and is not glycosylated,enzymatically deglycosylated huFcγ1-IL-7 protein, by treatment withPNGaseF, was evaluated. It was found to have similar activity to theuntreated form. Without wishing to be bound by theory, the somewhatdecreased activity of the fusion proteins may have been due not toglycosylation of the IL-7 moiety but instead to a steric effectresulting from a constrained N-terminus of the IL-7 moiety.

EXAMPLE 7 Pharmacokinetics of huFc-IL-7 Proteins

The pharmacokinetic (PK) profiles of an huFc-IL-7 fusion protein and ofrecombinant human IL-7 (Peprotech, Rocky Hill, N.J.) were evaluated, andthe results are depicted in FIG. 13. A single subcutaneous injection ofequimolar amounts of huFcγ2(h)(FN>AQ)-IL-7 or of recombinant human IL-7(50 micrograms) was administered to groups of C57BL6/J mice. Bloodsamples were obtained by retro-orbital bleeding at injection (i.e., att=0 min), and at 30 min, 1 hr, 2 hrs, 4 hrs, 8 hrs, 24, 48, 72, 96, 120and 144 hrs post-injection. Samples were collected in heparin-tubes toprevent clotting, and cells were removed by centrifugation in ahigh-speed Eppendorf microcentrifuge for 4 min at 12,500 g. PK valueswere calculated with the PK solutions 2.0™ software package (SummitResearch Services, Montrose, Colo.).

The concentration of administered IL-7 was determined in quadruplicateplasma samples at each time point by an ELISA specific for human IL-7.It was found that the pharmacokinetic behavior of the huFc-IL-7 andrecombinant IL-7 differed dramatically. For recombinant human IL-7, themaximum concentration (C_(max)) was 23.5 ng/ml at 2.0 hours postinjection (T_(max)), whereas for huFc-IL-7 C_(max) was 1588.7 ng/ml 24hours post injection. In addition, while recombinant human IL-7 wasabsorbed more rapidly than huFc-IL-7 (α-phase half-life 0.9 hours vs.12.4 hours), huFc-IL-7 was eliminated approximately 9-fold more slowlyfrom circulation during the β-phase. Thus, in terms of AUC (area underthe curve) as a measure of total drug exposure, mice receiving huFc-IL-7had a 572-fold higher exposure to the administered protein than micereceiving recombinant human IL-7. These data demonstrate a significantimprovement of huFc-IL-7 fusion proteins relative to free recombinanthuman IL-7 with regards to their PK. It was further found that the PKprofiles of huFc-IL-7 fusion proteins, such as huFcγ1-IL-7 andhuFcγ2(h)(FN>AQ)-IL-7, huFcγ1(YN>AQ)(linker2)-IL-7, andhuFcγ1(YN>AQ,d)(linker2)-IL-7, which were administered to the mice byintravenous injection, were similar to one another.

EXAMPLE 8 Efficacy of huFc-IL-7 in Lymphopenic Mice After Bone Marrow(BM) Transplantation

The efficacy of huFc-IL-7 fusion proteins compared to recombinant humanIL-7 was evaluated in vivo. For example, huFcγ2(h)(FN>AQ)-IL-7 orrecombinant human IL-7 (Peprotech, Rocky Hill, N.J.) was administered tolymphopenic mice after transplantation of T-cell-depleted bone marrow(BM), and the recovery of immune cell populations was assessed.

Essentially, recipient mice were lethally irradiated prior to BMtransplantation with two doses of 600 cGy total body irradiation at a 4hr interval, and BM cells re-suspended in PBS were infused into tailveins of recipient mice. At regular intervals from Day 5 onwards, anequimolar amount of huFc-IL-7 (7 μg) or recombinant human IL-7 (2.5 μg)(Peprotech, Rocky Hill, N.J.) was administered subcutaneously to therecipient mice. Over the course of the experiment, recipient mouse bloodsamples were taken, and lymphocyte cell concentrations in the sampleswere measured.

For BM cell transplantations, BM cells were obtained aseptically fromfemurs and tibias of BL/6.SJL (H2^(b), CD45.1) mice (Jackson Labs, BarHarbor, Me.) and depleted of T-cells by removing magnetically labeledT-cells over MACS® columns (Miltenyi Biotec, Auburn, Calif.). The degreeof T-cell depletion was monitored by FACS analysis with fluorescentlylabeled antibodies against CD45, αβ-TCR (T-cells) and 7-AminoActinomycin D (7-AAD, apoptotic cells) (Calbiochem, X). 10×10⁶ live(7-AAD-negative) BM cells (containing less than 1% T-cells) were usedper recipient mouse. In congeneic BM transplantations, B6 (H2^(b),CD45.2) mice were used as the recipient mouse strain, and in allogeneicBM transplantations B6C3F1 (H2^(b/k), CD45.2) mice were chosen.

Lymphocyte cell concentrations (as presented in Table 1) were measuredessentially as described by Brocklebank and Sparrow (Brocklebank andSparrow (2001) Cytometry 46:254). Briefly, fluorescent beads (TruCOUNT™Tubes, BD Biosciences, San Jose, Calif.) were dissolved in 40 μl of PBScontaining a mixture of lymphocyte-specific antibodies. Subsequently, 10μl of anti-coagulated blood was added, mixed and incubated for 30minutes in the dark at room temperature. Red blood cells were lysed in450 μl of Red Blood Cell lysis solution (BD Biosciences, San Jose,Calif.) and samples were analyzed by flow cytometry (BD FACSCalibur™, BDBiosciences, San Jose, Calif.). The concentration of a particularlymphocyte population (e.g. B-cells, T-cells or total leukocytes) wasdetermined by creating separate gates around lymphocytes and fluorescentbeads and reading the number of events within each gate. The number ofgated lymphocytes per microliter was calculated by dividing the numberof events in a gated lymphocyte region by the number of events in thegated bead region. This number was multiplied by the fraction of thenumber of beads per TruCOUNT™ tube (provided by the supplier) over thesample volume and finally multiplied by the sample dilution factor.

In one experiment, lymphocyte reconstitution was assessed in a congeneicBM transplantation setting, using materials and methods specified above.Recipient mice were injected with huFcγ2(h)(FN>AQ)-IL-7 at a dose of 7μg (125 μg IL-7/kg body weight), and lymphocyte cells were measured asdescribed. Donor lymphocytes were detected as CD45.1 positive cells,whereas endogenous lymphocytes of the recipient mice were detected asCD45.2 positive cells. Lymphocyte B-cells and T-cells were identifiedusing B220 and CD3 lymphocyte markers respectively. It was found that byDay 49, donor lymphocytes (CD45.1 positive cells) had repopulated therecipient mice to levels comparable to non-irradiated control mice,while endogenous lymphocytes (CD45.2 positive cells) had notsignificantly expanded. In addition, the treatment with the huFc-IL-7fusion protein did not cause significant toxicity. These resultsdemonstrated the efficacy of the fusion protein in expanding adoptivelytransferred lymphocyte populations. These results are depicted in FIG.16.

In another experiment, an allogeneic BM transplantation model, which maybetter simulate a clinical transplantation setting, was used to comparea huFc-IL-7 fusion protein to recombinant human IL-7, and the resultsare shown in Table 1. Again, methods and materials described above wereused. huFcγ2(h)(FN>AQ)-IL-7 and human IL-7 (equivalent of 125 μg IL-7/kgbody weight) were administered either every other day (q2d) or once aweek (q7d) from Day 5 to Day 56 after transplantation. PBS-treated donormice and irradiated, bone marrow recipient mice treated with PBS servedas controls.

In recipient mice treated with the fusion protein, donor-derived B-cells(CD45.1⁺, B220⁺, CD19⁺) reached baseline levels (as defined by the bloodconcentration of B-cells in donor control mice) 14 or 16 days aftertransplantation, when given q2d or q7d, respectively. In contrast, inrecipient mice treated with recombinant human IL-7, neither dosingregimen had an effect; B-cell numbers in PBS-treated and humanIL-7-treated recipient mice required about the same time to reachbaseline levels, about 28 days. In addition to accelerated B-cellreconstitution, huFc-IL-7 treatment promoted the continuous expansion ofB-cells until Day 33: huFc-IL-7 administered q2d resulted in a 7-foldincrease, whereas administered q7d resulted in a 2.5-fold increase inB-cell numbers compared to control mice. After Day 33, B-cell numbersdeclined, but were still approximately 2-fold higher than in controlmice. Also, after Day 33, levels of administered IL-7 proteins declinedin the blood, which partially may be due to the formation ofneutralizing antibodies to the human fusion protein. FIG. 14 representsthese results of B-cell reconstitution in irradiated, bone marrowtransplanted mice treated with recombinant human IL-7 and huFC-IL-7.

A similar result was observed regarding donor-derived T-cells (CD45.1⁺,CD3⁺, TCRαβ⁺). Treatment with the huFc-IL-7 fusion protein resulted inaccelerated T-cell reconstitution, whereas treatment with recombinanthuman IL-7 did not. Maximum T-cell levels were reached around Day 49.However, T-cell numbers above baseline (i.e., blood concentration ofT-cells in donor mice) were only achieved with a q2d dosing schedule ofthe huFc-IL-7 fusion protein, reaching about 1.5 fold the number ofT-cells in the control mice. FIG. 15 represents these results of T-cellreconstitution in irradiated bone marrow transplanted mice treated withrecombinant human IL-7 and huFc-IL-7.

Despite the transiently high numbers of donor B-cells and T-cells in therecipient mice under certain conditions, none of the experimental miceshowed any signs of morbidity during the course of the experiment.Analysis of internal organs at Day 55 did not reveal pathologicalabnormalities in liver, kidney, lung, spleen, thymus, lymph nodes,stomach, small intestine and colon. Thus, this allogeneictransplantation experiment demonstrated that the huFc-IL-7 fusionprotein was significantly superior in vivo over recombinant human IL-7in reconstituting lymphocytes after myeloablative conditioning.

TABLE 1 Effect of huFc-IL-7 fusion protein on immune cellreconstitution. Number of cells per microliter of blood Day Treatment 713 19 27 33 41 47 55 I. Donor Derived Leukocytes (CD45.1⁺) a.)Allogeneic PBS AVG 580.8 3576.8 4384.0 16805.6 20732.8 16395.2 21462.420329.6 BMT into B6C3 SEM 324.5 1717.0 1474.2 8367.6 10065.3 7997.311230.2 9693.2 mice huFc-IL-7, AVG 248.8 26597.0 130253.6 148048.0194266.4 168794.0 127848.0 79175.2 q2d SEM 89.3 4786.2 25106.8 18786.317131.1 25949.9 981.0 10740.2 huFc-IL-7, AVG 278.4 5848.8 32308.064256.0 74103.2 69042.4 55989.6 58519.2 q7d SEM 106.3 762.9 2786.36081.4 8345.9 14497.4 5989.8 6583.8 human IL- AVG 270.7 6020.0 6922.724278.7 37353.3 42166.7 33570.7 38138.7 7, q7d SEM 84.9 1229.2 1089.75235.3 2697.0 4691.4 764.5 3273.6 human IL- AVG 229.0 4603.0 4963.027443.0 27027.0 27797.0 32675.0 33205.0 7, q7d SEM 31.9 435.6 654.12513.8 1813.4 4084.6 6307.8 4894.7 b.) B6.SJL PBS AVG 30744.5 Controlmice SEM 3018.6 II. Donor Derived B-cells (CD45.1⁺CD19⁺B220⁺) a.)Allogeneic PBS AVG 32.0 1013.3 4257.3 19294.7 22549.3 17842.7 23841.322296.0 BMT into B6C3 SEM 5.7 25.7 661.1 2858.5 2625.4 1691.6 3783.71474.5 mice huFc-IL-7, AVG 16.0 17830.0 122122.4 131862.4 169745.6142972.0 98571.0 58661.6 q2d SEM 5.7 3755.4 24028.4 16974.5 15413.622834.6 2599.1 9352.9 huFc-IL-7, AVG 14.4 2559.0 28124.0 53573.6 59999.252932.8 41489.6 42899.2 q7d SEM 6.5 408.9 2464.1 4911.5 6337.2 10998.24821.1 5013.0 human IL- AVG 5.3 1550.7 4316.0 17802.7 28061.3 30177.324720.0 26104.0 7, q7d SEM 3.5 403.5 542.7 3689.5 1484.5 2861.6 633.82085.3 human IL- AVG 4.0 748.0 3179.0 19303.0 19203.0 17930.0 22460.021473.0 7, q7d SEM 1.6 56.1 443.3 1666.1 1429.5 2511.6 4458.6 3199.5 b.)B6.SJL PBS AVG 15572.6 Control mice SEM 1631.4 III. Donor Derived T-cells (CD45.1⁺CD3⁺abTCR⁺) a.) Allogeneic PBS AVG 0.0 206.7 192.0 1808.03928.0 4453.3 6178.7 5622.7 BMT into B6C3 SEM 0.0 121.6 46.2 245.0 546.7533.0 538.1 210.0 mice huFc-IL-7, AVG 0.0 124.0 839.0 5260.8 12252.815204.8 16628.0 13365.6 q2d SEM 0.0 24.9 155.5 1183.1 1734.8 1641.21524.5 1423.9 huFc-IL-7, AVG 0.0 38.4 228.0 2428.0 5492.0 9988.0 9252.88598.4 q7d SEM 0.0 6.1 38.5 326.2 918.1 2475.8 2748.6 1185.9 human IL-AVG 0.0 56.0 144.0 1057.3 3068.0 5368.0 5081.3 7112.0 7, q7d SEM 0.0 3.342.5 160.1 154.0 645.5 113.7 409.9 human IL- AVG 0.0 41.0 88.0 1493.02579.0 3918.0 5021.0 6043.0 7, q7d SEM 0.0 2.5 20.2 187.5 238.0 694.6724.2 1013.1 b.) B6.SJL PBS AVG 10832.0 Control mice SEM 1500.4

EXAMPLE 9 Efficacy of huFc-IL-7 on T-Cell Transplantations inLymphopenic Mice

The efficacy of huFc-IL-7 fusion proteins was also evaluated in a T-celltransplantation model. In essence, a homogeneous (clonal) population ofT-cells was transferred into immunodeficient, irradiated mice, therecipient mice were administered the huFc-IL-7 fusion protein, and thedegree of T-cell reconstitution and, eventually, T-cell function, wasassessed.

To obtain a homogeneous population of T-cells, splenocytes were takenfrom P14 TCR-tg/RAG mice (Charles River Laboratories, Wilmington,Mass.), which are devoid of B-cells. In addition, all T-cells of thesemice express the transgenic T-cell receptor (TCR), P14, which isspecific for a viral epitope (gp33 of LCMV).

Single cell suspensions of splenocytes were injected intravenously intothe tails of RAG Cγ^(−/−) immunodeficient mice (Charles RiverLaboratories) that had been irradiated once with 650 Rads (sub-lethaldose) 4 hrs prior to transplantation. On alternate days starting at Day2, recipient mice were administered 7 μg of the fusion proteinhuFcγ2(h)(FN>AQ)-IL-7. A control group of recipient mice wereadministered PBS. The degree of T-cell reconstitution in response to thehuFc-IL-7 fusion protein or PBS was determined by measuring the presenceof P14 T-cells (CD8⁺Vβ8.1⁺Vα2⁺ cells) in the blood by flow cytometry.

It was found that at Day 35, mice that were administered the huFc-IL-7fusion protein had a 17-fold increase in T-cell numbers (35,000cells/μl) compared to control mice (2,000 cells/μl). Indeed, the levelsof T-cell reconstitution exceeded those seen in untreated P14 TCR mice(23,000 cells/μl). In addition, in these huFc-IL-7-treated mice asignificant fraction of reconstituted T-cells had up-regulated theIL-2Rα receptor subunit, CD25, on the cell surface. Thus, not only wasthe huFc-IL-7 fusion protein useful in expanding transplanted T-cells,but in addition may have preconditioned the transferred T-cells to beresponsive to cytokines, such as IL-2.

EXAMPLE 10 huFc-IL-7 Adjuvant Therapy for Immunocommomised Patients

Numerous clinical settings are envisaged in which patients may benefitfrom huFc-IL-7 adjuvant therapy. For example, new treatment modalitiesare being developed for pediatric patients with malignant disease suchas lymphoblastic or myeloid leukemias, who, following a myeloablativetherapy, are treated by allogeneic hematopoietic stem celltransplantation to reconstitute the immune system.

To increase markedly the potential donor pool for these patients, it hasbeen found that G-CSF mobilized peripheral blood stem cells (PBSCs) frommatched unrelated donors or haplo-identical donors with 1-3 HLA locimismatches may be a source of cells, provided that the transplant isdepleted of T-cells (see Handgretinger et al. (2001) Annals NY Acad.Sciences 938:340-357). This depletion drastically reduced the occurrenceof acute graft-to-host transplant rejection (GvHD); however, it isbelieved that because of the low concentration of T-cells, there was asignificant delay in immunoreconstitution. Patients were at high risk ofviral infections for at least the first 6 month post transplant, andT-cells did not return to normal levels for a year (Handgretinger et al,(2001) Annals NY Acad. Sciences 938:340-357; Lang et al., (2003) Blood101:1630-6). Therefore, it would be advantageous to increase the rate ofrepopulation of T-cells and of other immune cells in these patients.

Patients that will benefit huFc-IL-7 therapy include patients with achildhood leukemia, such as a lymphoblastic leukemia or a myeloidleukemia. Children having this disorder will first undergo amyeloablative conditioning therapy which may be based either on thechemotherapeutic agent busulfan or total body irradiation combined withchemotherapy. For instance, according to the patient's diagnosis andage, the patient is treated with total body irradiation (typically 6treatments of 2 Gy each), rabbit anti-thymocyte globulin (10 mg/kg dailyfor 3 days), etoposide (40 mg/kg) and cyclophosphamide (120 mg/kg).

To obtain, CD34 positive (pos) stem cells for transplant, peripheralblood stem cells (PBSCs) of a histocompatible (allogeneic) donor aremobilized with a daily dose of 10 micrograms/kg of G-CSF for 6 days, andare harvested by leukapharesis on days 5 and 6. Generally, about20×10⁶/kg CD34 pos stem cells are obtained and transplanted. CD34 posstem cells are purified from the PBSCs by positive selection with anantiCD34 antibody in a SuperMACS system (Magnetic activated cellsorting, Miltenyi Biotec) and eluted. T-cell depletion is typicallyaround 5 logs, to about 10×10³ cells/kg. Aggregates and other debris areexcluded from the graft by FACS sorting. The cell suspension is infusedinto the patient via a central venous catheter. Optionally, the graftmay include purified populations of other immune cells, such ashaploidentical NK cells, DCs, monocytes, as well as CD34 neg stem cells.

To assess engraftment, an absolute neutrophil count is performed.Engraftment is considered successful once neutrophil levels remain above50 cells/microliter. Reconstitution of immune cells is monitored by FACSanalysis, weekly at first and, once T-cell recovery begins, every 3months.

To augment immunoreconstitution, the patient is treated with a huFc-IL-7fusion protein such as huFcγ2h(FN>AQ)-IL-7 orhuFcγ1(YN>AQ)(linker2)-IL-7. Approximately 3 weeks after transplant (orafter engraftment is established), the patient receives a subcutaneousadministration of huFcγ2h(FN>AQ)-IL-7 or huFcγ1(YN>AQ)(linker2)-IL-7 ata dose of about 1.5 mg/m² (or a dose in the range of 0.15 mg/m² to 15mg/m²), about 2 times a week for 6 months-12 months, until T-cell countsreach 50% of normal levels. It is found that the prognosis of thepatient is improved due to lowered risk of viral infection, one of themain post-transplant complications. It is further found that thistreatment does not significantly increase the risk of acute GvHD.

In addition to administration of huFc-IL-7 protein, other medicationsare optimally given prophylactically. These include, for example,acyclovir, metronidazole, flucanazole and co-trimoxazole. For the firstthree months, the patient may receive weekly administration ofimmunoglobulins, as well as of G-CSF.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A fusion protein comprising: a first portion comprising an Fc regionfrom IgG1 or IgG2 wherein the Fc region comprises a hinge region, a CH2domain, and a CH3 domain; and a second portion comprising a polypeptidehaving an amino acid sequence comprising at least 95% sequence identitywith wild-type mature human interleukin-7 (IL-7) wherein wild-typemature human IL-7 consists of amino acid residues 26-176 of SEQ ID NO:1,and wherein amino acid residues corresponding to positions 70 and 91 ofwild-type mature human IL-7 are glycosylated and the amino acid residuecorresponding to position 116 of wild-type mature human IL-7 isnon-glycosylated.
 2. The fusion protein of claim 1, wherein the aminoacid residue corresponding to position 116 of wild-type mature humanIL-7 is asparagine.
 3. The fusion protein of claim 1, wherein thepolypeptide having 95% sequence identity with wild-type mature humanIL-7 comprises disulfide bonding between residues corresponding topositions Cys2 and Cys92, Cys34 and Cys 129, and Cys47 and Cys 141 ofwild-type mature human IL-7.
 4. The fusion protein of claim 1, furthercomprising a linker between the first portion and the second portion. 5.The fusion protein of claim 1, wherein the Fc region is from IgG1. 6.The fusion protein of claim 5, wherein the Fc region further comprisesan amino acid substituted in place of Asn297at glycosylation site of theFc region.
 7. The fusion protein of claim 6, wherein the Fc regionfurther comprising an amino acid substituted in place of Tyr296 at theglycosylation site of the Fc region.
 8. The fusion protein of claim 7,wherein Ala is substituted in place of Tyr296 and Gln is substituted inplace of Asn297 at the glycosylation site of the Fc region.
 9. Thefusion protein of claim 1, wherein the Fc region is from IgG2.
 10. Thefusion protein of claim 9, wherein the Fc region further comprises anIgG1 hinge.
 11. The fusion protein of claim 9, wherein the Fc regionfurther comprises an amino acid substituted in place of Asn atglycosylation site of the Fc region.
 12. The fusion protein of claim 11,wherein the Fc region further comprises an amino acid substituted inplace of the Phe residue adjacent the Asn residue at the glycosylationsite of the Fc region.
 13. The fusion protein of claim 12, wherein Alais substituted in place of Phe and Gln is substituted in place of Asn atthe glycosylation site of the Fc region.
 14. The fusion protein of claim1, wherein said second portion comprises wild-type mature human IL-7consisting of amino acid residues 26-176 of SEQ ID NO:1, and wherein theamino acid residues corresponding to positions 70 and 91 of saidwild-type mature human IL-7 are glycosylated and the amino acid residuecorresponding to position 116 of said wild-type mature human IL-7 isnon-glycosylated.
 15. A purified nucleic acid encoding the fusionprotein of claim
 1. 16. A cultured host cell comprising the nucleic acidof claim
 15. 17. A fusion protein comprising: a first portion comprisingan Fc region from IgG1 or IgG2 wherein the Fc region comprises a hingeregion, a CH2 domain, and a CH3 domain; and a second portion comprisingan amino acid sequence that is at least 95% identical to amino acidresidues 26-176 of SEQ ID NO:1 (wild-type mature human IL-7) except thatresidues 121 to 138 of SEQ ID NO:1 are deleted in said second portion,and wherein the amino acid residues corresponding to positions 70 and 91of wild-type mature human IL-7 are glycosylated and the amino acidresidue corresponding to position 116 of wild-type mature human IL-7 isnon-glycosylated.